Detection method using both fluorescence and chemiluminescence labels

This application is a national stage of International Application PCT/US2021/073053, filed Dec. 21, 2021, which claims the priority of U.S. Provisional Application No. 63/129,946, filed Dec. 23, 2020. The contents of the above-identified applications are incorporated herein by reference in their entireties.

The invention relates to a method for detecting an analyte in a liquid sample using both fluorescent and chemiluminescent labels. The dual labels provide accurate quantitation on both high concentration and low concentration samples.

Antigen antibody pairs, receptor ligand pairs, and complementary nucleic acid pairs are often used as the basis of detection in diagnostic tests. The antibodies, complementary nucleic acids or receptors fixed on a substrate to capture the target in the sample are usually called capturing molecules. After capturing molecules bind to the target analyte in the sample, detection antibodies, nucleic acids or receptors are used to detect the captured target analyte. The detection antibody, nucleic acids or receptors may have fluorescence, chemiluminescence or electrochemiluminescence labels on them to provide a signal for detection.

Among the labels, fluorescent molecules are commonly used. However, the number of target molecules in a sample varies greatly in a diagnostic detection, and their concentrations vary at least 5,000 times; for some specific targets, the concentration varies even more than 300,000 times. Although a fluorescent label can effectively detect targets with a low-end concentration, the concentration of the fluorescent label may increase by several orders of magnitude when the target concentration is high. When fluorescence molecules are close to each other, self-quenching will occur, which leads to the nonlinear relationship between the fluorescence signal and the concentration of the target, and results in inaccurate quantitation.

Chemiluminescent label requires no external radiant energy for detection. The energy comes from photons released after the chemical reaction breaks the chemical bond. However, the energy conversion efficiency of chemiluminescence labels is not 100%. A lot of energy is dissipated by heat rather than generating photons. Chemiluminescence immunoassay often shows assay variation when the analyte concentration is low.

There is still a need for improved detection methods to solve the problems encountered in immunoassay's.

Terms used in the claims and specification are to be construed in accordance with their usual meaning as understood by one skilled in the art except and as defined as set forth below.

“About,” as used herein, refers to within +10% of the recited value.

An “analyte-binding molecule”, as used herein, refers to any molecule capable of participating in a specific binding reaction with an analyte molecule.

An “aspect ratio” of a shape refers to the ratio of its longer dimension to its shorter dimension.

A “binding molecular,” refers to a molecule that is capable to bind another molecule of interest.

A “binding pair,” as used herein, refers to two molecules that are attracted to each other and specifically bind to each other. Examples of binding pairs include, but not limited to, an antigen and an antibody against the antigen, a ligand and its receptor, complementary strands of nucleic acids, biotin and avidin, biotin and streptavidin, biotin and neutravidin (a deglycosylated version of avidin), lectin and carbohydrates. Preferred binding pairs are biotin and streptavidin, biotin and avidin, biotin and neutravidin, fluorescein and anti-fluorescein, digioxigenin/anti-digioxigenin, DNP (dinitrophenol)/anti-DNP.

“Chemiluminescence,” as used herein, refers to the emission of energy with limited emission of luminescence, as the result of a chemical reaction. For example, when luminol reacts with hydrogen peroxide in the presence of a suitable catalyst, it produces 3-aminophthalate in an excited state, which emits light when it decays to a lower energy level.

“Immobilized,” as used herein, refers to reagents being fixed to a solid surface. When a reagent is immobilized to a solid surface, it is either be non-covalently bound or covalently bound to the surface.

A “monolithic substrate,” as used herein, refers to a single piece of a solid material.

A “probe,” as used herein, refers to a substrate coated with a thin-film layer of analyte-binding molecules at the sensing side. A probe has a distal end and a proximal end. The proximal end (also refers to probe tip in the application) has a sensing surface coated with a thin layer of analyte-binding molecules. Preferably, the substrate has a low fluorescence background, and the substrate can be quartz, silicon, metal, ceramic or plastic.

The present invention improves a fluorescent detection method, for example, as described in US Publication No. 2011/0312105, for measuring a concentration of an analyte. The present method uses a chemiluminescent label in addition to a fluorescent label in the method. The fluorescent detection provides sensitivity and accuracy for low concentration analytes, and the chemiluminescent detection improves the accuracy of high concentration analytes.

In the detection method of the present invention, both fluorescence and chemiluminescence labels are used. A fluorescence signal is read first to obtain the advantages of low-end sensitivity and accuracy. Then a chemiluminescence signal is read. When the fluorescence signal is high and nearly saturated, the chemiluminescence signal provides an accurate high-end concentration of the target analyte.

First Aspect

In a first aspect, the present invention is directed to a method of detecting an analyte in a liquid sample. The method comprises the steps of: (a) dipping a probe tip in a sample solution to bind an analyte, if present, to a first antibody on the probe tip, wherein the probe having a first antibody immobilized on the tip of the probe; (b) dipping the probe tip into a reagent solution comprising a biotin-conjugated second antibody to form an immunocomplex among the analyte, the first antibody, and the second antibody on the probe tip, wherein the first antibody and the second antibody are two different antibodies each against the analyte; (c) dipping the probe tip in a wash solution; (d) dipping the probe tip into a solution comprising streptavidin labeled with a fluorescent marker and streptavidin labeled with a chemiluminescent marker; (e) dipping the probe tip in a read vessel and measuring the fluorescent signal of materials bound on the probe tip; (f) quantitate the analyte concentration based on the fluorescent signal against a first calibration curve; (g) dipping the probe tip to a triggering solution to generate a chemiluminescent signal of materials bound on the probe tip; (h) quantitate the analyte concentration based on the chemiluminescent signal against a second calibration curve, and (i) determining the analyte concentration based on either the fluorescent signal against the first calibration curve or based on the chemiluminescent signal against the second calibration curve, depending on the fluorescent signal of the analyte, using a pre-established cut-off value.

In step (a), the probe can be any shape such as rod, cylindrical, round, square, triangle, etc. In one embodiment, the probe has an aspect ratio of length to width of at least 5 to 1, or 10 to 1. A rod-shape is preferred.

The probe has a small tip for binding analytes. The tip has a smaller surface area with a diameter ≤5 mm, preferably ≤2 mm or ≤1 mm, e.g., 0.5-2 mm.

The probe tip is coated with a first antibody which binds to the analyte in a sample. Methods to immobilize reagents to the solid phase (the sensing surface of the probe tip) are common in immunochemistry and involve formation of covalent, hydrophobic or electrostatic bonds between the solid phase and reagent. The first antibody can be directly immobilized on the sensing surface. Alternatively, the first antibody can be indirectly immobilized on the sensing surface through a binding pair. For example, anti-fluorescein can be first immobilized either by adsorption to the solid surface or by covalently binding to aminopropylsilane coated on the solid surface. Then the first antibody that is labeled with fluorescein can be bound to the solid surface through the binding of fluorescein and anti-fluorescein (binding pair).

The probe tip is dipped into a sample vessel for 20 seconds to 60 minutes, preferably 20 seconds to 10 minutes, to bind the analyte to the first antibody on the probe tip. After step (a), the probe is optionally washed 1-5 times, preferably 1-3 times in a wash vessel containing a wash solution. This extra washing step may not be required because the amount of the carried-over solution is minimal due to a small binding surface area. The wash solution typically contains buffer and a surfactant such as Tween 20.

In step (b), the probe tip is dipped into a reagent vessel for 20 seconds to 10 minutes, preferably 20 seconds to 2 minutes to bind biotin-conjugated second antibody against the analyte to form an immunocomplex.

In Step (c), the probe is washed 1-5 times, preferably 1-3 times in a wash vessel containing a wash solution. The wash solution typically contains buffer and a surfactant such as Tween 20.

In step (d), two streptavidin reagents are used. i.e., streptavidin labeled with fluorescent labels and streptavidin labeled with chemiluminescent labels.

In one embodiment, each streptavidin is a monomer of streptavidin.

In another embodiment, each streptavidin reagent is further conjugated to a polymer. The polymer can be a polysaccharide (e.g., a copolymer of sucrose and epichlorohydrin (FICOLL®) or dextran), a polynucleotide, a dendrimer, a polyol, or polyethylene glycol. Polysaccharides in general exhibit negligible non-specific binding to many of the solid phase materials commonly employed in immunoassays. The polymer should have low non-specific binding, have greater than 400 or 500 kD in molecular weight to serve as an effective carrier of multiple binding streptavidins. FICOLL® is commercially available in 70K and 400K Dalton molecular weights. One preferred polymer is crosslinked FICOLL®. For example, a fluorescent streptavidin conjugate carries about 20 to 30 streptavidins per FICOLL® (2 million Daltons), and 2-3 Cy5 molecules per streptavidin. For example, a chemiluminescent streptavidin conjugate carries about 20 to 30 streptavidins per FICOLL® (2 million Daltons), and 2-3 acridinium esters molecules per streptavidin.

The size of the fluorescent label and chemiluminescent label is preferred to be small, having molecule weights ≤5000 Daltons: preferably ≤2000 Daltons: for example, 200-2000 daltons, or 300-1200 Daltons. A high molecular weight label when conjugated to streptavidin is likely to alter its biotin binding capacity and present steric hindrance.

The fluorescent label is selected from the group consisting of: cyanine, coumarin, xanthene and a derivative thereof. For example, the fluorescent dye is Cy5 (molecule weight MW 792), Alexa Fluor 647, DyLight 350 (MW 874), Dy Light 405 (MW793), DyLight 488 (MW 71011), Dy Light 550 (MW 982), Dy Light 594 (MW 1078), Dy Light 633 (MW 1066), Dy Light 650 (MW 1008), DyLight 680 (MW 950), Dy Light 755 (MW 1092), Dy Light 800 (MW 1050), an Oyster fluorescent dye, IRDye, or organic compounds comprising multiple rings chelated with a rare earth metal such as a lanthanide (Eu, Th, Sm, or Dy).

The chemiluminescent label is selected from the group consisting of: Ruthenium(II)tris-bipyridine (MW 1057), acridinium ester (9[[4-[3-[(2,5-dioxo-1-pyrrolidinyl)oxy]-3-oxopropyl]phenoxy]carbonyl]-10-methyl-acridinium trifluoromethane sulfonate, MW 632), and hemin (MW 652).

In each step (a)-(d), the reaction can be accelerated by agitating or mixing the solution in the vessel. For example, a lateral flow (orbital flow) of the solution across the probe tip can be induced, which accelerates the capture of target molecules by its binding partner immobilized to solid phase. For example, the reaction vessel can be mounted on an orbital shaker and the orbital shaker is rotated at a speed at least 50 rpm, preferably at least 200 rpm, more preferably at least 500 rpm, such as 500-1,000 rpm. Optionally, the probe tip can be moved up and down and perpendicular to the plane of the orbital flow, at a speed of 0.01 to mm/second, in order to induce additional mixing of the solution above and below the probe tip.

After step (d), the probe is washed 1-5 times, preferably 1-3 times in a wash vessel containing a wash solution, before reading the fluorescent signal (step (e)). To read a fluorescent label, the probe is placed in a clear-bottom well and read by a detector, such as those described in US 2011/0312105 (seeof the reference), or by the detector as shown inof this application.

In step (f), the analyte concentration is quantitated against a pre-established first calibration curve for fluorescent signals.

In steps (g) and (h), a chemiluminescent signal is generated, and the analyte concentration is quantitated against a second calibration curve, which is pre-established for chemiluminescent signals. For a chemiluminescent label, the probe is placed in a clear-bottom well containing a triggering solution having a co-reactant. For example, when the chemiluminescent label is Ruthenium(II)tris-bipyridine, the co-reactant is tripropylamine.

When the chemiluminescent label is acridinium ester, the co-reactants are (a) an aqueous solution containing HNOand HOin water, and (b) an aqueous solution containing NaOH and a cationic surfactant cetyltrimethylammonium chloride (CTAC). The light emitted is measured by a photomultiplier tube (PMT).

In step (i), the analyte concentration is determined based on either the first calibration curve (fluorescent signal) or the second calibration curve (chemiluminescent signal), depending on the fluorescent signal of the analyte, using a cut-off value, which is pre-established based on a fluorescent calibration curve. In general, when the analyte concentration is low, an accurate analyte concentration is determined based on the fluorescent signal and the fluorescent calibration curve. When the analyte concentration is high, an accurate analyte concentration is determined based on the chemiluminescent signal and the chemiluminescent calibration curve. When the analyte concentration is in the middle range, whether fluorescence or chemiluminescence is used to derive analyte concentration is based on a pre-established cutoff value of the fluorescence signal. Samples with fluorescence signals below the cutoff value will have the analyte concentration determined by the fluorescence calibration. Samples with fluorescence signals above the cutoff value will have the analyte concentration determined by the chemiluminescence calibration.

In the first aspect of the invention, biotin and streptavidin can be replaced by a first member of a binding pair and a second member of binding pair. A “binding pair” is defined in the Definitions. Biotin and streptavidin are a preferred binding pair. Other useful binding pairs are biotin and avidin, biotin and neutravidin, fluorescein and anti-fluorescein, digioxigenin/anti-digioxigenin, or DNP (dinitrophenol)/anti-DNP.

In one embodiment, the method comprises the steps of: (a) dipping a probe tip in a sample solution to bind an analyte, if present, to a first antibody on the probe tip, wherein the probe having a first antibody immobilized on the tip of the probe; (b) dipping the probe tip in a reagent solution comprising a second antibody conjugated to a first member of a binding pair to form a first immunocomplex among the analyte, the first antibody, and the second antibody on the probe tip, wherein the first antibody and the second antibody are two different antibodies each against the analyte; (c) dipping the probe tip in a wash solution; (d) dipping the probe tip in a solution comprising (i) a second member of binding pair labeled with a fluorescent label and (ii) the second member of binding pair labeled with a chemiluminescent label to form a second immunocomplex with the fluorescent label and a third immunocomplex with the chemiluminescent label on the probe tip; (e) dipping the probe tip in a read vessel and measuring the fluorescent signal of the second immunocomplex bound on the probe tip; (f) quantitate the analyte concentration based on the fluorescent signal against a first calibration curve; (g) dipping the probe tip to a triggering solution to generate a chemiluminescent signal from the third immunocomplex bound on the probe tip; (h) quantitate the analyte concentration based on the chemiluminescent signal against a second calibration curve, and (i) determining the analyte concentration based on either the first calibration curve or the second calibration curve, depending on the fluorescent signal of the analyte, using a pre-established cut-off value.

Second Aspect

In a second aspect of the invention, the method steps of detection are similar to the first embodiment except (i) the probe is pre-coated with an antibody against a hapten, and (ii) a sample, a first antibody conjugated with the hapten, and a second antibody conjugated with biotin are mixed before reacting with the probe tip.

In the second aspect, the present method comprises the steps of: (a) mixing a solution comprising a sample, a first antibody conjugated with a hapten, a second antibody conjugated with biotin, wherein the first antibody and the second antibody are two different antibodies each against the analyte; (b) dipping a probe tip into the solution of (a) to form an immunocomplex among the analyte, the first antibody, and the second antibody on the probe tip; (c) dipping the probe tip in a wash solution; (d) dipping the probe tip in a solution comprising streptavidin labeled with a fluorescent label and streptavidin labeled with a chemiluminescent label; (e) dipping the probe tip in a read vessel and measuring the fluorescent signal of materials bound on the probe tip; (f) quantitate the analyte concentration based on the fluorescent signal against a first calibration curve; (g) dipping the probe tip to a triggering solution to generate a chemiluminescent signal of materials bound on the probe tip; (h) quantitate the analyte concentration based on the chemiluminescent signal against a second calibration curve, and (i) determining the analyte concentration based on either the fluorescent signal against the first calibration curve or the chemiluminescent signal against a second calibration curve, depending on the fluorescent signal of the analyte, using a pre-established cut-off value.

Steps (c)-(i) of the second aspect of the invention are identical or similar to those described in the first aspect.

In the second aspect of the invention, biotin and streptavidin can be replaced by a first member of a binding pair and a second member of binding pair, respectively. A “binding pair” is defined in the Definitions. Biotin and streptavidin are a preferred binding pair. Other useful binding pairs are biotin and avidin, biotin and neutravidin, fluorescein and anti-fluorescein, digioxigenin/anti-digioxigenin, or DNP (dinitrophenol)/anti-DNP.

In one embodiment, the method comprises the steps of: (a) mixing a solution comprising a sample, a first antibody conjugated with a hapten, a second antibody conjugated with a first member of a binding pair, wherein the first antibody and the second antibody are two different antibodies each against the analyte; (b) dipping a probe tip into the solution of (a) to form a first immunocomplex among the analyte, the first antibody, and the second antibody on the probe tip; (c) dipping the probe tip in a wash solution; (d) dipping the probe tip in a solution comprising (i) a second member of binding pair labeled with a fluorescent label and (ii) the second member of binding pair labeled with a chemiluminescent label, to form a second immunocomplex with the fluorescent label and a third immunocomplex with the chemiluminescent label on the probe tip; (e) dipping the probe tip in a read vessel and measuring the fluorescent signal of the second immunocomplex bound on the probe tip; (f) quantitate the analyte concentration based on the fluorescent signal against a first calibration curve; (g) dipping the probe tip to a triggering solution to generate a chemiluminescent signal from the second immunocomplex bound on the probe tip; (h) quantitate the analyte concentration based on the chemiluminescent signal against a second calibration curve, and (i) determining the analyte concentration based on either the first calibration curve or the second calibration curve, depending on the fluorescent signal of the analyte, using a pre-established cut-off value.

Detection Device

is the structure diagram of the detection device that reads both fluorescence and chemiluminescence labels. The detection device comprises an optical module, which comprises a laser, a photon counter, a lens, a movable filter and a numerical aperture system. The fluorescent signal and chemiluminescent signal of the emitted light are detected by photomultiplier tubes (PMT). For measuring chemiluminescent signal, the laser is turned off and the filter and the numerical aperture system are removed. Then concentrated luminescence trigger A and trigger B are added into the reading cell with the substrate pump, and the chemiluminescence signal is generated.

The invention is illustrated further by the following examples that are not to be construed as limiting the invention in scope to the specific procedures described in them.

Quartz probes, 1 mm diameter and 2 cm in length, were coated with aminopropylsilane using a chemical vapor deposition process (Yield Engineering Systems, 1224P) following manufacturer's protocol. The probe tip was then immersed in a solution of murine monoclonal anti-fluorescein (Biospacific), 10 μg/ml in PBS (phosphate-buffered saline) at pH 7.4. After allowing the antibody to adsorb to the probe for 20 minutes, the probe tip was washed in PBS.